Activation and participation of cardiac progenitor cells (CPCs) in regeneration are critical for effective repair in the wake of pathologic injury. The process of stem cell activation and commitment involves increased energy demand and activation of mitochondrial biogenesis. To transition from a non-contracting progenitor cell requiring little energy to a beating cardiomyocyte requires development of an energetic infrastructure capable of supporting the high metabolic demands of the myocyte. However, the regulation of mitochondrial biology, signaling to regulate mitochondrial biogenesis, and the correlation between mitochondrial function and CPC aging are essentially unknown. The central hypotheses to be tested in this project are that mitochondria play an essential role in CPC maintenance, survival and differentiation. Furthermore, the hostile milieu in the injured heart interferes with pathways that regulate mitochondrial function in CPCs and impairs their regenerative capacity. In aim #1, we will demonstrate that mitochondrial biogenesis and function are critical for self-renewal and differentiation of CPCs. Experiments wiil address if upon commitment to a cardiac lineage, CPCs activate mitochondrial biogenesis concomitant with increased energy production via mitochondrial oxidative phosphorylation. Since mitochondria are a major source of reactive oxygen species, experiments will also examine if there is an increase in the CPCs' anti-oxidant defense system. In aim #2, we will show that mitochondrial biogenesis is regulated by intrinsic signaling and influenced by the external environment. Specifically, expenments will delineate the roles of AMPK and G- protein coupled receptor signaling pathways in regulating PGC-1 a, mitochondrial biogenesis, and differentiation in CPCs. Mitochondria are also known to accumulate mutations in their DNA with age but how this affects CPC function is currently unknown. Accordingly in aim #3, we will test the hypothesis that mitochondrial DNA damage impairs CPC function and blunts potential for myocardial repair. Using a mouse model carrying a proofreading defective mitochondrial DNA polymerase, we will explore the functional consequences of accumulating mtDNA mutations in CPCs in vitro and in vivo. These studies will provide novel and important insights into the molecular mechanisms of mitochondrial maturation integral to the execution of a cardiac differentiation program. These studies will also provide important new knowledge into why endogenous CPCs have compromised function and survival in the injured heart, which can be used to enhance their regenerative capacity for future therapies. RELEVANCE (See instructions): Heart failure is the leading a cause of morbidity and mortality in the United States. These studies will provide important and novel insights into the role of mitochondria in myocardial regenerative biology with the ultimate goal of restoration of myocardial healing.